Gephyrin promotes autonomous assembly and synaptic localization of GABAergic postsynaptic components without presynaptic GABA release.

Synapses containing γ-aminobutyric acid (GABA) constitute the primary centers for inhibitory neurotransmission in our nervous system. It is unclear how these synaptic structures form and align their postsynaptic machineries with presynaptic terminals. Here, we monitored the cellular distribution of several GABAergic postsynaptic proteins in a purely glutamatergic neuronal culture derived from human stem cells, which virtually lacks any vesicular GABA release. We found that several GABAA receptor (GABAAR) subunits, postsynaptic scaffolds, and major cell-adhesion molecules can reliably coaggregate and colocalize at even GABA-deficient subsynaptic domains, but remain physically segregated from glutamatergic counterparts. Genetic deletions of both Gephyrin and a Gephyrin-associated guanosine di- or triphosphate (GDP/GTP) exchange factor Collybistin severely disrupted the coassembly of these postsynaptic compositions and their proper apposition with presynaptic inputs. Gephyrin-GABAAR clusters, developed in the absence of GABA transmission, could be subsequently activated and even potentiated by delayed supply of vesicular GABA. Thus, molecular organization of GABAergic postsynapses can initiate via a GABA-independent but Gephyrin-dependent intrinsic mechanism.

Multiple N-linked glycosylation sites critically modulate the synaptic abundance of neuroligin isoforms.

In recent years, elegant glycomic and glycoproteomic approaches have revealed an intricate glycosylation profile of mammalian brain with enormous spatial and temporal diversities. Nevertheless, at a cellular level, it is unclear how these post-translational modifications affect various proteins to influence crucial neuronal properties. Here, we have investigated the impact of N-linked glycosylation on neuroligins (NLGNs), a class of cell-adhesion molecules that play instructive roles in synapse organization. We found that endogenous NLGN proteins are differentially glycosylated across several regions of murine brain in a sex-independent but isoform-dependent manner. In both rodent primary neurons derived from brain sections and human neurons differentiated from stem cells, all NLGN variants were highly enriched with multiple N-glycan subtypes, which cumulatively ensured their efficient trafficking to the cell surface. Removal of these N-glycosylation residues only had a moderate effect on NLGNs' stability or expression levels but particularly enhanced their retention at the endoplasmic reticulum. As a result, the glycosylation-deficient NLGNs exhibited considerable impairments in their dendritic distribution and postsynaptic accumulation, which in turn, virtually eliminated their ability to recruit presynaptic terminals and significantly reduced NLGN overexpression-induced assemblies of both glutamatergic and GABAergic synapse structures. Therefore, our results highlight an essential mechanistic contribution of N-linked glycosylations in facilitating the appropriate secretory transport of a major synaptic cell-adhesion molecule and promoting its cellular function in neurons.

Induction of synapse formation by de novo neurotransmitter synthesis.

A vital question in neuroscience is how neurons align their postsynaptic structures with presynaptic release sites. Although synaptic adhesion proteins are known to contribute in this process, the role of neurotransmitters remains unclear. Here we inquire whether de novo biosynthesis and vesicular release of a noncanonical transmitter can facilitate the assembly of its corresponding postsynapses. We demonstrate that, in both stem cell-derived human neurons as well as in vivo mouse neurons of purely glutamatergic identity, ectopic expression of GABA-synthesis enzymes and vesicular transporters is sufficient to both produce GABA from ambient glutamate and transmit it from presynaptic terminals. This enables efficient accumulation and consistent activation of postsynaptic GABAA receptors, and generates fully functional GABAergic synapses that operate in parallel but independently of their glutamatergic counterparts. These findings suggest that presynaptic release of a neurotransmitter itself can signal the organization of relevant postsynaptic apparatus, which could be directly modified to reprogram the synapse identity of neurons.